DNA origami has been a versatile technique in nucleic acid nanotechnology. However, the current DNA origami procedure is inconvenient for the construction of complex structures, which requires extensive pipetting. This would be problematic for the production of complex DNA nanostructures and limit the versatility of DNA origami.
Motivated by the necessity of a simpler and faster procedure, our proposed method alleviates the need of tedious pipetting in making a DNA nanostructure. Instead of synthesizing hundreds or thousands of staple strands, we used a single long DNA strand that combines of the required staple sequences, which then be cut into the staples. Our procedure enables a faster and more precise construction of complex DNA nanostructures.
Since its introduction in 2006, DNA origami has brought major advancement in DNA nanotechnology. The technique allows easy designing and construction of 2D and 3D nanostructures by making use of DNA’s self-assembly property. Its simple principle of “folding” a single DNA strand (the scaffold) with the help of shorter oligonucleotides (the staples) quickly attracted the attention of nanotechnologists. Although it’s initially performed for aesthetic purposes or proof-of-concept researches, applications of DNA origami structures rapidly emerged. Now, DNA origami has been widely utilized to create various functional nanostructures with ever-increasing complexity.
However, production of complex structures using the current DNA origami procedure face significant challenges. Larger and more complex DNA origami structures require a large amount of staple strands. With the current method, each sequence of staple strands need to be synthesized separately before being mixed with the scaffold strands. For structures with hundreds of staple sequences, the synthesis process would be troublesome since many different kinds of oligonucleotides are required. Furthermore, the current method also requires thermal annealing reactions, which takes a lot of time to produce the desired structures. Therefore, the application of DNA origami technique to build complex structures is currently limited. In order to efficiently build complex DNA nanostructures, a new method to perform DNA origami is necessary.
We propose a novel idea of preparing DNA origami staple strands that avoids the need of creating multiple different staples and does not require a thermal cycler. Instead of synthesizing each staple sequence separately, we combined all of them into a single monolithic DNA strand. The strand consists of the required staple sequences spaced by restriction sites. Combining all of the staples into one long strand alleviates the need of extensive pipetting.
In our project, we utilized three types of enzyme to aid with the preparation of staples: restriction enzyme, nicking endonuclease, and polymerase. Restriction enzyme cuts our monolithic strand into the component staple strands in the mixture. Afterwards, the resulting staple strands are amplified with the help of nicking endonuclease and polymerase through strand displacement amplification, which is an isothermal process (i.e. does not require thermal cycling). Combining all of the enzymes in the scaffold and monolithic strand mixture would produce the staple strands that will readily anneal with the scaffolds. In other words, aside from reducing the need of pipetting, our method also provides a one-step reaction of producing a DNA origami structure.
The relevance of our method lies in its ability to build complex DNA origami nanostructures with relative ease. Compared to the conventional DNA origami procedure, our method allows a convenient and quick construction of large and intricate nanostructures. In this regard, our method could revolutionize DNA nanotechnology by enabling the creation of DNA nanostructures that previously hard to build.
The procedure we outlined provides a feasible means of creating complicated DNA nanostructures. Compared to the conventional method, our procedure could be carried out faster since it combines the staple amplification and annealing procedures into a single reaction. To produce a complex structure with hundreds of staples, our proposed method is more feasible compared to the current DNA origami method.
Our proposed method offers a simple and elegant solution for building large and complicated DNA nanostructures. In our procedure, the separate synthesis of multiple DNA staples is no longer needed; it is replaced by cutting a long DNA strand into the required staples. The utilization of strand displacement amplification also renders thermal cycling unneeded. Thus, we simplified the procedure of DNA origami into an elegant one-step reaction.
We envision a future where DNA origami is the leading force in the field of nanotechnology. To achieve that goal, a simple and effective procedure of building DNA nanostructures with DNA origami is necessary. We believe our proposed method could go beyond the limitations of current techniques and pave a way to the creation of intricate nanostructures with beneficial functions.